Molecular markers in plant systematics and population biology

1
Molecular markers in plant systematics and
population biology

1. Introduction overview of the methods

Tomáš Fér tomas.fer_at_centrum.cz
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What are molecular markers ?

• information about organism obtained from analysis
  of its molecules proteins, DNA
• marker information unit targeted or randomly
  chosen part of the total information
• markers tells about genetic similarity (kinship)
  of individuals, populations or species

3
Marker examples

• information about enzyme molecule (e.g., its
  charge and mobility)
• sequence of nucleotides in DNA chain
• length of DNA fragment

4
Aspects of molecular data

• give an information about individual genotype
• information not dependent on environment
  conditions (no plasticity)
• assumption about selective neutrality i.e., no
  influence on individual fitness
• non-coding DNA regions
• same functionality of all isozymes
• qualitative information fragment presence,
  allele, nucleotide
• unique information about organism clone
  identification
• subject to modification during generative
  reproduction recombination

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Deposition of genetic information

• nucleus
• different ploidy level
• recombination
• biparental transfer

• plastids
• 1 circular molecule
• without recombination
• uniparental transfer

• mitochondrion
• structurally complicated
• common restructuring
• uniparental transfer

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Genome characteristics
nDNA animals plants cpDNA mtDNA animals plants
heritability biparental biparental angiosperms maternal, conifers paternal maternal maternal
structure linear linear circular circular circular, complex
size (kb) 4.9 x 104 7.0 x 108 5.0 x 104 3.0 x 108 71 214 15 20 200 2400
substitution rate 3.5 x 10-9 4.1 5.7 x 10-9 0.86 1.20 x 10-9 56 x 10-9 0.36 0.50 x 10-9
substitution rate relative to plant mtDNA 8.1 11.4 2.4 130.2 1.0
foreign sequences common common rare rare common
structural mutations common common rare rare common
recombination yes yes intramolecular no inter- and intramolecular
Lowe et al. 2004
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Molecular markers overview

• proteins isozymes
• DNA markers
• RFLP (Restriction Fragment Length Polymorphism)
• PCR based analysis of DNA fragments
• order of nucleotides DNA sequences
• whole genome analysis fragment length
  polymorphism
• RAPD (Random Amplified Polymorphic DNA)
• AFLP (Amplified Fragment Length Polymorphism)
• ISSRs (Inter Simple Sequence Repeats)
• information from specific genome regions
• PCR-RFLP (Polymerase Chain Reaction RFLP)
• microsatellites (Simple Sequence Repeats SSRs)
• SSCP (Single Strain Conformation Polymorphism)
• whole genome markers SNP, whole genome
  sequencing

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Isoenzymes (isozymes, allozymes)

• proteins catalysing basic biochemic reactions
• extracted from living tissues preferably leaves
• individual molecules (alleles)
  electrophoretically separated according to
  differences in mobility (electric charge)
• visualisation by color reactions

limited variation
codominant marker
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Isoenzyme gel example
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RFLPRestriction Fragment Length Polymorphism

• DNA je specifically cleaved to fragments by
  restriction endonuclease
• electrophoresis length separation
• large amount of fragments specific part is
  visualized by hybridisation with labelled probe
  (Southern blotting) e.g., visualization of
  cpDNA only
• variability insertion/deletion or mutation in
  restriction site

highly reproducible pattern labelled
probes required
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RFLP gels examples
http//www.ufpe.br/biolmol/Tec-mol-biol/RFLP-real.
JPG
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PCR-RFLP

• amplification of specific DNA region using PCR
  with two specific primers
• use of consensual primers (e.g., cpDNA)
• applicable to almost all plant species
• restriction of amplified region by different
  restriction enyzmes
• electrophoresis of fragments

universal method lower variability when cpDNA
is used
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tRFLP terminal - Restriction Fragment Length
Polymorphism
http//hpl.umces.edu/faculty/bcrump/TRFLP.pdf
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RAPDRandom Amplified Polymorphic DNA

• fragments are generated using PCR with one
  arbitrary primer (decanucleotide)
• electrophoretic separation of fragments according
  to their length
• polymorphism is caused by
• mutation in the place where primers anneal
  (priming site)
• insertion/deletion in the amplified DNA

simple method results difficuilt to
reproduce, uncomparable between labs
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RAPD gel example
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AFLPAmplified Fragment Length Polymorphism

• combination of RFLP and subsequent PCR of
  selected fragments
• DNA restriction by two different enzymes
• selective amplification of a subset of fragments
• fluorescence visualization of fragments on the
  gel (using automated sequencer)

highly polymorfic, reflects variability of
whole genome high reproducibility,
reliability dominant marker homo- a
heterozygotes cannot be distinguished
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AFLP Amplified Fragment Length Polymorphism
Mueller Wolfenbarger (1999), TREE
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AFLP gel example
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MicrosatellitesSSRs simple sequence repeats

• tandem repeats of several nucleotides

AGGCTATATATATAGGCA 1 AGGCTATATATA--GGCA 2

• alelles differ by number of repeats

codominant marker, highly variable
relationships among alleles can be assesed
necessity to evolve primers for study species
20
Example of microsatellite analysis
http//www.qub.ac.uk/bb-old/prodohl/gel/Images/mul
tiplex_set_1b.gif
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Microsatellites - homozygotes and
heterozygotes(Nuphar lutea)
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ISSRs Inter Simple Sequence Repeats

• length variation of regions between
  microsatellite loci
• primer microsatellite sequence

variable marker, simple dominant marker
J. Košnar, unpubl.
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SSCP Single Strand Conformation Polymorphism

• method for finding unknown point mutation
• PCR amplification of target region
• denaturation electrophoresis of ssDNA
• mutation changes tertiary structure
  (conformation) of the chain and thus its mobility
  in gel

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SSCP gel example
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DNA sequencing

• determination of the sequence of nucleotides in
  DNA chain
• use of automated sequencers fluorescence base
  labelling
• specific primers for PCR amplification of the
  target region

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Next generation sequencing NGSmasivelly
parallel sequencing

• several platforms
• 454 (Roche) pyrosequencing
• Illumina (Solexa)
• SOLiD (ABI)

• diverse approaches/applications
• shotgun sequencing
• amplicon sequencing

principle of pyrosequencing
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Sequencing

• coding genes conservative
• systematics at the level of families, genera
  (rbcL)
• spacers, introns variable regions
• systematics at the level of genera, species and
  below(trnL-trnF, atpB-rbcL)

• chloroplast genes
• rbcL
• atpB
• matK

• nuclear genes
• ITS
• 18S rDNA
• 26S rDNA

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Chloroplast DNA
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Types of molecular markersimportant differences

• variability high low

high low microsatellites allozymes AFLP chl
oroplast markers
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Types of molecular markersimportant differences

• variability high low

• heritability dominant codominant

dominant codominant AFLP allozymes RAPD mic
rosatellites RFLP (PCR-RFLP)
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Types of molecular markersimportant differences

• variability high low

• heritability dominant codominant

• recombination yes no

yes ne nuclear markers organelar (cp,
mt)(diploid, polyploid) (haploid)
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Types of molecular markersimportant differences

• variability high low

• heritability dominant codominant

• recombination yes no

• transfer to next gener. biparental uniparental

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Types of molecular markersimportant differences

• variability high low

• heritability dominant codominant

• recombination yes no

• transfer to next gener. biparental uniparental

• mutation rate high low

high low microsatellites allozymes introns,
spacers exons
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Which factors influence markers?
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Utility of markers in different types of studies
RFPL and PCR-RFLP RFPL and PCR-RFLP RFPL and PCR-RFLP RFPL and PCR-RFLP Sequencing Sequencing Sequencing Sequencing
Allozymes nDNA cpDNA mtDNA (plant) mtDNA (animal) RAPD AFLP SSR nDNA cpDNA mtDNA (plant) mtDNA (animal)
Genetic diversity
Population differentiation
Gene flow () () () ()
Polyploidy - - - - - -
Hybridization
Phylogeny () () - - () ()
Individual genotyping () - - - - - -
Phylogeography () ? () - - () ()
excellent () has been used good - unlike
ly to be usefull or useless OK ? uncertain or
not used
based on Lowe et al. 2004
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Literature

• Avise J.C. (2004) Molecular markers, natural
  history and evolution.
• Baker A.J. (2000) Molecular methods in ecology.
• Beebee T. Rowe G. (2004) An introduction to
  molecular ecology.
• Caetano-Anollés G. Gresshoff P.M. (1998) DNA
  markers. Protocols, applications, and overviews.
• Henry R.J. (2001) Plant genotyping. The DNA
  fingerprinting of plants.
• Karp A. et al. (1998) Molecular tools for
  screening biodiversity.
• Lowe A., Harris S. Ashton P. (2004) Ecological
  Genetics Design, Analysis, and Application.
• Weising K. et al. (2005) DNA fingerprinting in
  plants. Principles, methods, and applications.
• DeSalle R. Schierwater (1998) Molecular
  Approaches to Ecology and Evolution.
• Karp A. et al. (1996) Molecular techniques in
  the assesment of botanical diversity. Annals of
  Botany 78143-149
• Ouborg N.J. et al. (1999) Population genetics,
  molecular markers and the study of dispersal in
  plants. J. Ecol. 87551-568.
• Parker G.P. et al. (1998) What molecules can
  tell us about populations Choosing and using a
  molecular marker. Ecology 79 361-382
• Vekemans X. Jacquemart (1997) Perspectives on
  the use of molecular markers in plant population
  biology. Belg. J. Bot. 12991-100

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Molecular markers in botany

1. (4.10.) molecular markers characteristics,
   differences, technique overview
2. (18.10.) molecular markers overview of
   applications and questions
3. (25.10.) isozymes electrophoresis, evaluation
   of codominant data, population genetics
4. (1.11.) DNA - structure, PCR techniques,
   applications, RAPD
5. (8.11.) AFLP principle, applications,
   evaluation of dominant data
6. (22.11.) restriction techniques (RFLP,
   PCR-RFLP), cpDNA, phylogeography
7. (29.11.) microsatellites nuclear, chloroplast,
   isolation, data evaluation, applications
8. (13.12.) sequencing chloroplast DNA, gene and
   non-coding regions
9. (20.12.) sequencing II nuclear DNA, rDNA, ITS,
   low-copy markers
10. (3.1. 2013) "new" and other methods real-time
    PCR, next-generation sequencing, and
    applications...
11. (10.1.) Methods and applications summary, other
    use of molecular markers
12. (TBA) student presentations, exam

http//botany.natur.cuni.cz/fer/markers/indexE.htm